1. Field of the Invention
The invention relates to diagnostic methods to determine disease state, nutritional state, or effect of therapy by measurement of one or more unbound metabolites by measuring a fluorescence change upon binding to one or more probes. Preferably, the probes are fluorescently labeled muteins of intracellular lipid binding proteins (iLBP).
2. Description of the Related Art
Metabolomics is advancing rapidly as a result of new technologies and the expanding interest in systems biology (Goodacre, R. (2005) Metabolomics—the way forward, Metabolomics 1, 1-2) This advance is being driven by the recognition that physiologic phenotype is essentially a reflection of the metabolic profile, and therefore, metabolic profiling should provide an accurate representation of the states of health and disease. The activity of a given metabolite is frequently dictated by its solubility as a “free” or unbound molecule in aqueous bodily fluids. For many metabolites the unbound concentration represents a small fraction of the total, with most of the total metabolite bound in carrier complexes. Total metabolite concentrations are typically measured, but it is the unbound metabolite that interacts with targets such as protein receptors and cell membranes (for example, Sorrentino, D., et al. (1989) At physiologic albumin/oleate concentrations oleate uptake by isolated hepatocytes, cardiac myocytes, and adipocytes is a saturable function of the unbound oleate concentration, J. Clin. Invest. 84, 1325-1333). A profiling of unbound metabolite concentrations should therefore provide the most accurate measure of physiologic health.
For example, profiling unbound rather than total metabolites is especially relevant for the long chain free fatty acids (FFA), which play major roles in signaling, macromolecular structure and energy production. Long chain FFA are sparingly soluble, yet act through extra- and intra-cellular aqueous phases to bind to target macromolecules (Sorrentino, et al. supra; Cupp, D., et al. (2004) Fatty acid:albumin complexes and the determination of long chain free fatty acid transport across membranes, Biochemistry 43, 4473-4481; Kampf, J. P. et al. (2004) Fatty acid transport in adipocytes monitored by imaging intracellular FFA levels, J. Biol. Chem. 279, 35775-35780). In many instances, FFA-mediated signaling events can be abolished by adding fatty-acid-free bovine serum albumin, which reduces the unbound FFA (FFAu) concentration without changing the total FFA concentration (Poitout, V. (2003) The ins and outs of fatty acids on the pancreatic beta cell, Trends Endocrinol. Metab 14, 201-203; Kleinfeld, A. M, et al. (2005) Free fatty acid release from human breast cancer tissue inhibits cytotoxic T lymphocyte-mediated killing, J. Lipid Res. 46, 1983-1990). Total serum concentrations of long chain FFA are in the millimolar range while FFA-protein receptor binding affinities are in the nanomolar range as are FFAu concentrations (Kampf, J. P., et al. supra; Richieri, G. V., et al. (1993) Interactions of long chain fatty acids and albumin: Determination of free fatty acid levels using the fluorescent probe ADIFAB, Biochemistry 32, 7574-7580; Richieri, G. V., et al. (1994) Equilibrium constants for the binding of fatty acids with fatty acid binding proteins from intestine, heart, adipose, and liver; measured with the fluorescence probe ADIFAB, J. Biol. Chem. 269, 23918-23930; Apple, F. S., et al. (2004) Unbound Free Fatty Acid Concentrations Are Increased in Cardiac Ischemia, Clinical Proteomics 1, 41-44). The hydrophobic nature of FFA and low FFAu concentrations have made it difficult to measure FFAu in biological fluids, and thus, total FFA is typically measured even though FFA-dependent signaling events are triggered by unbound rather than total FFA concentrations.
More than 40 different species of FFA with widely different biological activities have been identified in human serum (Yli-Jama, P., et al. (2002) Serum free fatty acid pattern and risk of myocardial infarction: a case-control study, J Intern. Med. 251, 19-28). Striking examples of their different biological activities include the induction of apoptosis in various cell types by palmitate (16:0) but not oleate (18:1) (de Vries, J. E., et al. (1997) Saturated but not mono-unsaturated fatty acids induce apoptotic cell death in neonatal rat ventricular myocytes, J. Lipid Res. 38, 1384-1394; Listenberger, L. L., et al. (2001) Palmitate-induced apoptosis can occur through a ceramide-independent pathway, J. Biol. Chem. 276, 14890-14895; Hickson-Bick, D. L., et al. (2002) Palmitate-induced apoptosis in neonatal cardiomyocytes is not dependent on the generation of ROS, Am. J. Physiol Heart Circ. Physiol 282, H656-H664) and the inhibition of cytotoxic T lymphocyte signaling by oleate (OA) but not palmitate (PA) (Kleinfeld, et al. supra; Richieri, G. V. et al. (1989) Free fatty acid perturbation of transmembrane signaling in cytotoxic T lymphocytes, J. Immunol. 143, 2302-2310). In addition, alterations in the profile of total plasma FFA have been reported in association with disease states (Yli-Jama, P., et al. supra; Lorentzen, B., et al. (1995) Fatty acid pattern of esterified and free fatty acids in sera of women with normal and pre-eclamptic pregnancy, Brit. J. Obstetrics and Gynecology 102, 530-537; Rodriguez de Turco, E. B., et al. (2002) Systemic fatty acid responses to transient focal cerebral ischemia: influence of neuroprotectant therapy with human albumin, J Neurochem. 83, 515-524; Yli-Jama, P., et al. (2002) Serum non-esterified very long-chain PUFA are associated with markers of endothelial dysfunction, Atherosclerosis 164, 275-281; Freedman, S. D., et al. (2004) Association of cystic fibrosis with abnormalities in fatty acid metabolism, N. Engl. J. Med. 350, 560-569).
Plasma FFAu levels are a reflection of the FFA-albumin binding equilibrium. The affinities of albumin for different FFA can differ by more than 2 orders of magnitude (Richieri, G. V., et al. (1993) supra; Spector, A. A. (1975) Fatty acid binding to plasma albumin, J. Lipid Res. 16, 165-179), and therefore, the equilibrium FFAu profile will differ from the total profile. FFAu profiles have not been reported previously because none of the available measurement techniques can resolve the nanomolar quantities of individual FFAu in the FFAu mixtures present in aqueous biological fluids. However, measurements of individual FFAu and/or average values for FFAu mixtures have been carried out previously using the acrylodan labeled fatty acid binding proteins ADIFAB and ADIFAB2 (Apple, et al. supra; Richieri, G. V. et al. (1995) Unbound free fatty acid levels in human serum, J. Lipid Res. 36, 229-240).